Method for culturing mammalian cells to improve recombinant protein production

ABSTRACT

The present invention relates to methods for mammalian cell culture. The methods make use of independent tyrosine and cystine feed streams.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/112,202 filed Feb. 24, 2014, which is a national stageapplication under 37 U.S.C. §371 of International Application No.PCT/US2012/034532 filed Apr. 20, 2012 which claims the benefit under 35U.S.C. §119 of U.S. Provisional Application Ser. No. 61/477,900, filedApr. 21, 2011 and U.S. Provisional Application Ser. No. 61/490,981,filed May 27, 2011, which are hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to a method for mammalian cell culture.The method makes use of independent tyrosine and cystine feed streams.

BACKGROUND OF INVENTION

Clinical manufacture of therapeutic proteins is an expensive, largescale endeavor. Maintaining cell growth and viability throughout thecell culture process is very important. As the demand for greaterquantities of therapeutic recombinant proteins increases, positiveincreases in protein production, cell growth and viability are soughtout by implementing new methods to improve cell development, mediaoptimization and process control parameters, and to intensify harvestand purification processes. Much effort is now being placed on processoptimization, particularly methods and formulations for feedingproduction cell cultures. One such method is the use of a concentratedfeed medium during the production phase, often used in fed batch cultureprocesses, to improve protein titer, cell growth, and/or cell viability.

Tyrosine is a critical amino acid for sustaining cultured cells ingrowth and viability and is included in growth media and concentratedproduction media formulations. A depletion of tyrosine in a productionculture can lead to decreases in cell growth, viability and/or proteinproduction.

Due to poor solubility at neutral pH, tyrosine cannot be compounded athigh concentrations in either the growth or concentrated feed medium.Therefore, only a limited amount of tyrosine can be compounded intomedia formulations without causing the media to precipitate.Phenylalanine can convert to tyrosine via the enzyme phenylalaninehydroxylase (PAH) in certain cell types and can also be included inmedia formulations.

Cysteine is another important amino acid for sustaining culturedmammalian cells. However, cysteine readily oxidizes to form cystine inneutral or slightly alkaline solutions. So while cysteine is freelysoluble in water, it may contribute to insolubility and/or precipitationwhen in its oxidized form.

Mammalian cells are typically grown in cultures that are at or nearneutral pH, such as from about pH 6.5 to about pH 7.5. Tyrosine andcysteine, necessary components of mammalian cell culture media, cancause media formulations to destabilize in the neutral conditionsnecessary for cell growth.

New cell culture media formulations and/or methods for feedingproduction cultures that provide even incremental improvements in cellgrowth, viability and/or protein production are valuable, given theexpense of large scale cell culture processes and the difficulty andexpense of building and obtaining regulatory approval for newlarge-scale, commercial culture facilities.

There is a continuing need to develop media formulations and feedingmethods that are able to provide adequate amino acid levels in aproduction culture to maintain and improve cell viability, specificproductivity, and titer. Any improvements to cell culture mediaformulations and/or feeding strategies that allow for flexibility andcustomization to facilitate desirable recombinant polypeptideexpression, titer, cell growth and/or cell viability can lead to ease ofmaintenance, higher production levels, thereby reducing the costsassociated with manufacturing protein therapeutics. The inventionfulfills these needs by providing a simple, easy and inexpensive methodof increasing cell growth and protein production.

SUMMARY OF THE INVENTION

The present invention provides a method of culturing Chinese HamsterOvary (CHO) cells expressing a recombinant protein, comprising growingthe CHO cells in a defined serum-free culture medium during a growthphase and maintaining the CHO cells in the cell culture medium during aproduction phase by supplementing the cell culture with a concentrateddefined serum-free feed medium that does not contain tyrosine, cysteineor cystine, and further supplementing the cell culture with anindependent tyrosine and cystine feed, wherein viability was prolonged,specific productivity and titer was maintained and not diminished by theindependent tyrosine and cystine feed. In one embodiment the independenttyrosine and cystine feed provides at least about 0.1 mM to at leastabout 2.0 mM tyrosine at each feed. In a related embodiment theindependent tyrosine and cystine feed provides at least about 1.38 mMtyrosine. In another embodiment the independent tyrosine and cystinefeed provides at least about 0.17 mM to at least about 0.72 mM cystineat each feed. In a related embodiment the independent tyrosine andcystine feed provides at least about 0.50 mM cystine. In anotherembodiment the independent tyrosine and cystine feed begins at least byday 5 of the production phase. In a related embodiment the independenttyrosine and cystine feed begins on day 3 of the production phase. Inanother embodiment the independent tyrosine and cystine feed beginsprior to the production phase. In yet another embodiment the independenttyrosine and cystine feed is made concurrently with the feed of theconcentrated serum-free defined feed medium. In another embodiment theindependent tyrosine and cystine feed is not concurrent with the feed ofthe concentrated serum-free defined feed medium. In a further embodimentthe recombinant protein is selected from the group consisting of a humanantibody, a humanized antibody, a chimeric antibody, a recombinantfusion protein, or a cytokine.

The invention also provides a method of culturing CHO cells expressing arecombinant protein, comprising growing the CHO cells in a definedserum-free culture medium during a growth phase and maintaining the CHOcells in the cell culture medium during a production phase bysupplementing the cell culture with a concentrated serum-free definedfeed medium containing tyrosine and further supplementing the cellculture with an independent tyrosine feed, wherein viability wasprolonged, specific productivity was maintained, and titer was improvedcompared to CHO cells not receiving independent tyrosine feeds. Withinone related embodiment the independent tyrosine provides at least about1 mM and 2 mM tyrosine at each feed. In yet another related embodimentthe independent tyrosine feed provides at least about 1 mM tyrosine.Within another related embodiment the concentration of tyrosine in thecell culture medium does not exceed 8 mM. In yet another relatedembodiment the independent tyrosine feeds begin just prior to theproduction phase. Within another related embodiment the independenttyrosine feeds begin on day 7. In yet another related embodiment theindependent tyrosine feed is made concurrently with the feed of theconcentrated serum-free defined feed medium. Within certain otherrelated embodiments the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph showing tyrosine concentration over time, FIG. 1B isa graph showing specific productivity (Qp) over time, FIG. 1C is a graphshowing titer over time, FIG. 1D is a graph showing viability over time,FIG. 1E is a graph showing phenylalanine concentration over time.Symbols: (□) Concentrated feed media only, no independent tyrosinefeeds. Low tyrosine levels coincided with a drop in viability and Qpfrom day 9 onwards. Phenylalanine accumulated over time and did notconvert to tyrosine after tyrosine depletion.

FIG. 2A is a graph showing titer over time, FIG. 2B is a graph showingspecific productivity (Qp) over time, FIG. 2C is a graph showingviability over time, FIG. 2D is a graph showing cell diameter over time,FIG. 2E is a graph showing tyrosine concentration over time, FIG. 2F isa graph showing phenylalanine concentration over time. Symbols: dashed(□) Cells provided with concentrated feed media only (control), solid(▪) Cells were provided independent feeds of a concentrated feed mediaand a tyrosine solution (1 mM). Arrows indicate timing of tyrosineaddition. Without tyrosine depletion, titer was enhanced, specificproductivity maintained, viability prolonged and cell diameter increasedfrom day 9 onwards. Phenylalanine concentration remained the same.

FIG. 3A is a graph showing titer over time, FIG. 3B is a graph showingspecific productivity (Qp) over time, FIG. 3C is a graph showingviability over time, FIG. 3D is a graph showing cell diameter over time,FIG. 3E is a graph showing tyrosine concentration over time, FIG. 3F isa graph showing phenylalanine concentration over time. Symbols: dashed(×) Cells provided with independent feeds of a concentrated feed mediaand a tyrosine solution (1 mM feed) starting on day 7 (control), solid(●) cells provided with independent feeds of a concentrated feed mediaand a tyrosine solution (1.5 mM feed), solid (♦) cells provided withindependent feeds of a concentrated feed media and a tyrosine solution(2 mM feed), and solid (▴) cells provided with independent feeds of aconcentrated feed media and a tyrosine solution (4 mM feed). Arrowsindicate timing of tyrosine additions. Tyrosine concentration did notaffect the phenylalanine concentration. Extension of culture durationresulted in increasingly higher titer from day 16 onwards.

FIG. 4A is a graph showing viable cell density over time, FIG. 4B is agraph showing viability over time, FIG. 4C is a graph showing titer overtime. Symbols: solid (♦) Concentrated feed media containing cysteine butnot tyrosine. Supplemented with independent tyrosine feeds. Open (⋄)Concentrated feed media that does not contain cysteine or tyrosine.Supplemented with independent cysteine and independent tyrosine feeds.Growth, viability and antibody productivity were not diminished as aresult of removing tyrosine and cysteine from the feed medium anddelivering them as a separate concentrated feeds.

FIG. 5A is a graph showing viable cell density over time, FIG. 5B is agraph showing viability over time, FIG. 5C is a graph showing titer overtime, FIG. 5D is a graph showing lactate over time. In all cases theconcentrated feed media did not contain tyrosine, cysteine or cystine.Symbols: solid (♦) Supplemented with independent feeds of a combinedtyrosine and cystine feed solution. Open (⋄) Supplemented withindependent cysteine and independent tyrosine feeds. Growth, viability,antibody productivity and lactate production were not diminished as aresult of removing tyrosine and cysteine from the feed medium anddelivering them as a separate concentrated feeds.

DETAILED DESCRIPTION OF THE INVENTION

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. Unlessotherwise noted, the terms “a” or “an” are to be construed as meaning“at least one of”. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described. The methods and techniques described hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates (1992), and Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990). All documents, or portions ofdocuments, cited in this application, including but not limited topatents, patent applications, articles, books, and treatises, are herebyexpressly incorporated by reference.

The invention is based on the discovery that use of an independent feedof a concentrated tyrosine solution was able to overcome the depletionof tyrosine in the culture medium when the culture only receivedconcentrated production medium feeds that contained tyrosine in additionto other amino acids such as phenylalanine as well as other nutrients.Addition of the independent tyrosine feed increased cell growth,viability and polypeptide production from a recombinantly engineeredanimal cell line expressing a protein of interest, compared to culturesreceiving concentrated feed medium alone. Addition of the independenttyrosine feed offset the tyrosine depletion seen in cultures receivingonly the concentrated feed medium and resulted in enhanced culturerobustness and improved the yield of the polypeptide of interest.

The present invention provides a method of culturing Chinese HamsterOvary (CHO) cells expressing a recombinant protein. The inventive methodcomprises growing the CHO cells in a defined serum-free culture mediumduring a growth phase followed by maintaining the CHO cells during aproduction phase, wherein the cell culture medium is periodicallysupplemented with a concentrated feed medium and is further supplementedwith periodic independent tyrosine feeds, wherein viability wasprolonged, specific productivity was maintained, and titer was improvedcompared to CHO cells not receiving independent tyrosine feeds. Theconcentration of tyrosine added by each independent tyrosine feed is atleast about 1 mM-2 mM, preferably about 1 mM. The formulation of theconcentrated feed medium contains up to about 4.5 mM tyrosine. Theindependent tyrosine feeds are adjusted such that the concentration oftyrosine in the culture medium does not exceed 8 mM. The independenttyrosine feeds can begin just prior to or at the start of the productionphase. The independent tyrosine feed can be accomplished by fed batch tothe cell culture medium on the same or different days as theconcentrated feed medium. Such independent tyrosine feeds can be addedto the cell culture medium after one or more days, and can also be addedrepeatedly during the course of the production phase, as long astyrosine depletion in the cell culture medium is avoided. For example,the production phase can last from 7 days to as long as 8, 9, 10, 11,12, 13, or 14 days or longer. The cell culture medium can besupplemented with the independent tyrosine feeds immediately and/or ondays 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and/or later days of theproduction phase.

The invention also provides the use of an independent concentrated feedsolution containing tyrosine and cystine. It was discovered thatindependent concentrated feed solution containing tyrosine and cystinewas able to adequately supplement a mammalian cell culture receivingfeeds of a concentrated feed medium that did not contain tyrosine,cysteine or cystine in its formulation. Independent feeds of thetyrosine and cystine solution maintained cell growth, viability andpolypeptide production from a recombinantly engineered mammalian cellline expressing a protein of interest, without negative impact on thecell culture or the protein production. The combined cystine andtyrosine feed solution can be prepared at high pH so the feed solutionhas the added benefit of not requiring subsequent viral inactivatingtreatments such as pasteurization or viral filtration. The pH of thefeed solution is at least about pH 10.0 to at least about pH 12.0. ThepH of the feed solution can be 10.0, 10.5, 11.0, 11.5, 12.0 or any valuein between. Preferably the pH is at least about 10.0. Removing cysteineand tyrosine from the concentrated feed medium allows for the reductionor removal of stabilizers, such as sodium pyruvate (Published USApplication No. 2009/0123975), in concentrated feed medium formulationswithout the resulting cell culture media instability due toprecipitation of cysteine and tyrosine. Independent feeds of aconcentrated cystine and tyrosine feed solution did not causeprecipitation in the culture medium.

The present invention provides a method of culturing Chinese HamsterOvary (CHO) cells expressing a recombinant protein. The inventive methodcomprises growing the CHO cells in a defined serum-free culture mediumduring a growth phase followed by maintaining the CHO cells in the cellculture medium during a production phase, wherein the cell culturemedium is periodically supplemented with a concentrated definedserum-free feed medium that does not contain tyrosine, cysteine orcystine in its formulation and is further supplemented with periodicindependent feeds of a tyrosine and cystine feed solution, whereinviability was prolonged, specific productivity and titer was maintainedand not diminished by the independent tyrosine and cystine feed. Theconcentration of tyrosine added by each independent tyrosine and cystinefeed is at least about 0.1 mM to at least about 2.0 mM tyrosine. Theconcentration of tyrosine is at least about 0.1 mM, 0.5 mM, 1.0 mM, 1.25mM, 1.3 mM, 1.5 mM, 1.75 mM, 2.0 mM or any value in between. Preferablythe tyrosine concentration is at least about 1.38 mM. The concentrationof cystine added by each independent tyrosine and cystine feed is atleast about at least about 0.17 mM to at least about 0.72 mM. Theconcentration of tyrosine is at least about 0.17 mM, 0.2 mM, 0.03 mM,0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.72 mM and any value in between.Preferably the cystine concentration is at least about 0.5 mM. Theindependent tyrosine and cystine feed can begin prior to or at the startof the production phase. The independent tyrosine and cystine feed canbe accomplished by fed batch to the cell culture medium on the same ordifferent days as the concentrated feed medium. Such independenttyrosine and cystine feeds can be added to the cell culture medium afterone or more days, and can also be added repeatedly during the course ofthe production phase, as long as tyrosine, cysteine and cystinedepletion in the cell culture medium is avoided. For example, theproduction phase can last from 7 days to as long as 8, 9, 10, 11, 12,13, or 14 days or longer. The cell culture medium can be supplementedwith the independent tyrosine and cystine feed during the growth stageand/or on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or later duringthe production phase.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. Polypeptides can be of scientific or commercialinterest, including protein-based drugs. Polypeptides include, amongother things, antibodies, fusion proteins, and cytokines. Peptides,polypeptides and proteins are produced by recombinant animal cell linesusing cell culture methods and may be referred to as “recombinantpeptide”, “recombinant polypeptide” and “recombinant protein”. Theexpressed protein(s) may be produced intracellularly or secreted intothe culture medium from which it can be recovered and/or collected.

Examples of polypeptides that can be produced with the methods of theinvention include proteins comprising amino acid sequences identical toor substantially similar to all or part of one of the followingproteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391),erythropoeitin, thrombopoeitin, calcitonin, IL-2, angiopoietin-2(Maisonpierre et al. (1997),Science 277(5322): 55-60), ligand forreceptor activator of NF-kappa B (RANKL, WO 01/36637), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-CSF,Australian Patent No. 588819), mast cell growth factor, stem cell growthfactor (U.S. Pat. No. 6,204,363), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_00682; Rüegg andPytela (1995), Gene 160:257-62) growth hormone, insulin, insulinotropin,insulin-like growth factors, parathyroid hormone, interferons includinga-interferons, γ-interferon, and consensus interferons (U.S. Pat. Nos.4,695,623 and 4,897471), nerve growth factor, brain-derived neurotrophicfactor, synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon,interleukins, colony stimulating factors, lymphotoxin-β, leukemiainhibitory factor, and oncostatin-M. Descriptions of proteins that canbe produced according to the inventive methods may be found in, forexample, Human Cytokines: Handbook for Basic and Clinical Research, allvolumes (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge,Mass., 1998); Growth Factors: A Practical Approach (McKay and Leigh,eds., Oxford University Press Inc., New York, 1993); and The CytokineHandbook, Vols. 1 and 2 (Thompson and Lotze eds., Academic Press, SanDiego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. Nos. 5,395,760 and5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EP PatentNo. 0460846, U.S. Pat. Nos. 4,968,607, and 5,767,064,), IL-1 receptorantagonists (U.S. Pat. No. 6,337,072), IL-1 antagonists or inhibitors(US Patent Nos. 5,981,713, 6,096,728, and 5,075,222) IL-2 receptors,IL-4 receptors (EP Patent No. 0 367 566 and U.S. Pat. No. 5,856,296),IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK, WO01/36637 and U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and4), and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be producedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified, including human,humanized, chimeric, multi-specific, monoclonal, polyclonal, andoligomers or antigen binding fragments thereof Also included areproteins having an antigen binding fragment or region such as Fab, Fab′,F(ab′)₂, Fv, diabodies, Fd, dAb, maxibodies, single chain antibodymolecules, complementarity determing region (CDR) fragments, scFv,diabodies, triabodies, tetrabodies and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33,CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2,IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF,TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No. 6,235,883) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC1, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, HER-3, a tumor-associatedglycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes thatare present in elevated levels in the sera of patients with colon and/orpancreatic cancer, cancer-associated epitopes or proteins expressed onbreast, colon, squamous cell, prostate, pancreatic, lung, and/or kidneycancer cells and/or on melanoma, glioma, or neuroblastoma cells, thenecrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4,B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphlycoccus aureus. Specific examples of known antibodieswhich can be produced using the methods of the invention include but arenot limited to adalimumab, bevacizumab, infliximab, abciximab,alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

The invention can also be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Håkansson et al.(1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fc portionof an antibody such as etanercept (a p75 TNFR:Fc), and belatacept(CTLA4:Fc).

For the purposes of this invention, cell culture medium is a mediasuitable for growth of animal cells, such as mammalian cells, in invitro cell culture. Cell culture media formulations are well known inthe art. Typically, cell culture media are comprised of buffers, salts,carbohydrates, amino acids, vitamins and trace essential elements. Thecell culture medium may or may not contain serum, peptone, and/orproteins. Various tissue culture media, including serum-free and definedculture media, are commercially available, for example, any one or acombination of the following cell culture media can be used: RPMI-1640Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM),Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove'sModified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium,and serum-free media such as EX-CELL™ 300 Series (JRH Biosciences,Lenexa, Kans.), among others. Cell culture media may be supplementedwith additional or increased concentrations of components such as aminoacids, salts, sugars, vitamins, hormones, growth factors, buffers,antibiotics, lipids, trace elements and the like, depending on therequirements of the cells to be cultured and/or the desired cell cultureparameters.

Cell culture media may be serum-free, protein-free, and/or peptone-free.“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. “Protein-free” applies to cellculture media free from exogenously added protein, such as transferrin,protein growth factors IGF-1, or insulin. Protein-free media may or maynot contain peptones. “Peptone-free” applies to cell culture media whichcontains no exogenous protein hydrolysates such as animal and/or plantprotein hydrolysates. Eliminating serum and/or hydrolysates from cellculture media has the advantage of reducing lot to lot variability andenhancing processing steps, such as filtration. However, when serumand/or peptone are removed from the cell culture media, cell growth,viability and/or protein expression may be diminished or less thanoptimal. As such, serum-free and/or peptone-free cell culture medium maybe highly enriched for amino acids, trace elements and the like. See,for example, U.S. Pat. Nos. 5,122,469 and 5,633,162. Although there aremany media formulations, there is a need to develop defined mediaformulations that perform as well or preferably better than thosecontaining animal sera and/or peptones.

Defined cell culture media formulations are complex, containing aminoacids, inorganic salts, carbohydrates, lipids, vitamins, buffers andtrace essential elements. Identifying the components that are necessaryand beneficial to maintain a cell culture with desired characteristicsis an on going task. Defined basal media formulations which aresupplemented or enriched to meet the needs of a particular host cell orto meet desired performance parameters is one approach to developingdefined media. Identifying those components and optimum concentrationsthat lead to improved cell growth, viability and protein production isan ongoing task.

By cell culture or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, and operated in a batch, fedbatch, continuous, semi-continuous, or perfusion mode are available formammalian cell culture.

Mammalian cells, such as CHO cells, may be cultured in small scalecultures, such as for example, in 100 ml containers having about 30 mlof media, 250 ml containers having about 80 to about 90 ml of media, 250ml containers having about 150 to about 200 ml of media. Alternatively,the cultures can be large scale such as for example 1000 ml containershaving about 300 to about 1000 ml of media, 3000 ml containers havingabout 500 ml to about 3000 ml of media, 8000 ml containers having about2000 ml to about 8000 ml of media, and 15000 ml containers having about4000 ml to about 15000 ml of media. Large scale cell cultures, such asfor clinical manufacturing of protein therapeutics, are typicallymaintained for days, or even weeks, while the cells produce the desiredprotein(s).

During this time the culture can be supplemented with a concentratedfeed medium containing components, such as nutrients and amino acids,which are consumed during the course of the production phase of the cellculture. Concentrated feed medium may be based on just about any cellculture media formulation. Such a concentrated feed medium can containmost of the components of the cell culture medium at, for example, about5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×,600×, 800×, or even about 1000× of their normal amount. Concentratedfeed media are often used in fed batch culture processes.

Fed batch culture is a widely-practiced culture method for large scaleproduction of proteins from mammalian cells. See e.g. Chu and Robinson(2001), Current Opin. Biotechnol. 12: 180-87. A fed batch culture ofmammalian cells is one in which the culture is fed, either continuouslyor periodically, with a concentrated feed medium containing nutrients.Feeding can occur on a predetermined schedule of, for example, everyday, once every two days, once every three days, etc. The culture can bemonitored for tyrosine, cystine and/or cysteine levels in the culturemedium and can be adjusted through feedings of a concentrated tyrosineor tyrosine and cystine solution so as to keep tyrosine, cysteine and/orcystine within a desired range. When compared to a batch culture, inwhich no feeding occurs, a fed batch culture can produce greater amountsof protein. See e.g. U.S. Pat. No. 5,672,502.

The method according to the present invention may be used to improve theproduction of recombinant proteins in both single phase and multiplephase culture processes. In a single phase process, cells are inoculatedinto a culture environment and the disclosed methods are employed duringthe single production phase. In a multiple stage process, cells arecultured in two or more distinct phases. For example cells may becultured first in one or more growth phases, under environmentalconditions that maximize cell proliferation and viability, thentransferred to a production phase, under conditions that maximizeprotein production. In a commercial process for production of a proteinby mammalian cells, there are commonly multiple, for example, at leastabout 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases that occur indifferent culture vessels preceding a final production phase. The growthand production phases may be preceded by, or separated by, one or moretransition phases. In multiple phase processes, the method according tothe present invention can be employed at least during the productionphase, although it may also be employed in a preceding growth phase. Aproduction phase can be conducted at large scale. A large scale processcan be conducted in a volume of at least about 100, 500, 1000, 2000,3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. A growth phasemay occur at a higher temperature than a production phase. For example,a growth phase may occur at a first temperature from about 35° C. toabout 38° C., and a production phase may occur at a second temperaturefrom about 29° C. to about 37° C., optionally from about 30° C. to about36° C. or from about 30° C. to about 34° C. In addition, chemicalinducers of protein production, such as, for example, caffeine,butyrate, and hexamethylene bisacetamide (HMBA), may be added at thesame time as, before, and/or after a temperature shift. If inducers areadded after a temperature shift, they can be added from one hour to fivedays after the temperature shift, optionally from one to two days afterthe temperature shift.

The invention finds particular utility in improving or maintaining cellgrowth, viability and/or protein production via cell culture processes.The cell lines (also referred to as “host cells”) used in the inventionare genetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, and/or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells and/or cell lines to express a polypeptide of interest are wellknown to those of skill in the art; for example, various techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring LaboratoryPress, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990,pp. 15-69.

Animal cell lines are derived from cells whose progenitors were derivedfrom a multi-cellular animal. One type of animal cell line is amammalian cell line. A wide variety of mammalian cell lines suitable forgrowth in culture are available from the American Type CultureCollection (Manassas, Va.) and commercial vendors. Examples of celllines commonly used in the industry include VERO, BHK, HeLa, CV1(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1),PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells arewidely used for the production of complex recombinant proteins, e.g.cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J.Biol Chem 263:6352-6362; McKinnonet al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J.Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines becausethe efficient DHFR selectable and amplifiable gene expression systemallows high level recombinant protein expression in these cells (KaufmanR. J. (1990), Meth Enzymol 185:537-566). In addition, these cells areeasy to manipulate as adherent or suspension cultures and exhibitrelatively good genetic stability. CHO cells and proteins recombinantlyexpressed in them have been extensively characterized and have beenapproved for use in clinical commercial manufacturing by regulatoryagencies.

The methods of the invention can be used to culture cells that expressrecombinant proteins of interest. The expressed recombinant proteins maybe produced intracellularly or be secreted into the culture medium fromwhich they can be recovered and/or collected. In addition, the proteinscan be purified, or partially purified, from such culture or component(e.g., from culture medium or cell extracts or bodily fluid) using knownprocesses and products available from commercial vendors. The purifiedproteins can then be “formulated”, meaning buffer exchanged, sterilized,bulk-packaged, and/or packaged for a final user. Suitable formulationsfor pharmaceutical compositions include those described in Remington'sPharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton,Pa.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

EXAMPLES Example 1

It was observed that when growing CHO cells expressing recombinantantibodies in a chemically defined media containing tyrosine, thattyrosine was depleted during the production process despite regularsupplements to the culture medium with a concentrated feed mediumcontaining tyrosine. The depletion resulted in a fast drop in viability,decrease in specific productivity (Qp) and loss of titer, whichnegatively impacted the production of the recombinant protein. (FIG. 1A,B, C, D). Phenylalanine accumulated in the culture medium but did notconvert to tyrosine after tyrosine depletion (FIG. 1E)

Tyrosine is a poorly soluble amino acid and as such it is difficult toincrease the amount of tyrosine in the concentrated feed medium beyond4.5 mM or 1.2 g/L to meet the demands of the production culture.Phenylalanine is known to convert tyrosine via PAH but did notcontribute tyrosine to the tyrosine depleted cell culture medium. In anattempt to overcome the rapid depletion of tyrosine during theproduction, a concentrated tyrosine solution was fed to the cell culturemedium in addition to the concentrated feed medium that includedtyrosine in its formulation.

CHO cells expressing a recombinant antibody were inoculated at 1×10⁶c/ml by centrifugation in 120 ml of a serum-free defined growth mediumcontaining 0.6 g/L tyrosine. Cultures were maintained in vented 500 mlshake flasks and kept in a 36° C., 5% CO₂.

A 150 g/L tyrosine 2Na⁺ 2H₂O concentrated tyrosine feed solution wasprepared in dH₂O (SAFC Biosciences Lenexa, Kans.).

Production cultures were maintained for 16 days. Flasks were sampled (14mls) on days 5, 7, 9, 11, 13, and 16. Cultures were fed 9% based oncurrent volume (9-10.5 mls) of a concentrated serum-free defined feedmedium containing 1.2 g/L tyrosine, up to day 13. Glucose was maintainedat a range of 6-8 g/L and 1 mM tyrosine 2Na⁺ 2H₂O supplements (0.15-0.2mls) using the concentrated tyrosine solution were independently addedstarting on day 7 or 9 and subsequent feed days. A day 0 sample and pre-and post-feed samples up to day 16 were saved for titer and amino acidanalysis. A culture that did not receive the independent tyrosine feedswas included as a control and to confirm initial observations oftyrosine depletion.

Viable cell density (VCD) and viability were measured by the CEDEX(Innovatis, Germany) and metabolites by the NOVA BioProfile 100+ (NOVABiomedical, Mass.). Values of pH, pO₂, and pCO₂ were analyzed by theBioprofile pHox (NOVA Biomedical, Mass.) and osmolality by the model2020 osmometer (Advanced Instruments, Norwood, Mass.). Titer wasmeasured by reverse-phase HPLC analysis using affinity chromatographywhere Protein A was immobilized on a column support. At neutral pH,antibody molecules were bound to the Protein A through the Fc regionwhile host-cell proteins, conditioned media components and buffer wereeluted from the column in the flow-through. Captured antibodies wereeluted at acidic pH and detected by UV absorbance at 280 nm. Acalibration curve was derived from a universal antibody standard and thecorresponding peak areas using linear regression analysis.Concentrations of the antibody in the test samples were then calculatedfrom the calibration curve and the ratio of the extinction coefficientsfrom the Universal antibody standard and the antibody tested.

Amino acid analysis was performed using an AccuTag pre-columnderivatization chemistry reagent kit according to the manufacturer'sinstructions (Waters Corporation, Milford, Mass.)

The culture that received the supplemental tyrosine feeds achieved atiter of 5.2 g/L in 16 days compared to 4.1 g/L observed in the control(FIG. 2A). Specific productivity was maintained, viability was prolongedand cell diameter increased as a result of the additional tyrosine.Viability was 80% on day 16 in the culture receiving the additionaltyrosine compared to 60% without supplemented tyrosine and cell diameterincreased from 18 μm to 20 μm respectively in the cells cultured withsupplemental tyrosine (FIGS. 2B to 2D). Amino acid analysis did not showtyrosine depletion in the cultures receiving independent tyrosine feeds,indicating that these cells received sufficient levels even thoughtyrosine was being heavily consumed (FIG. 2E). The control cultureshowed tyrosine depletion and phenylalanine was not used to synthesizeadditional tyrosine in response to the depletion (FIG. 2F). Lactatelevels were similar between the cultures; however the ammonia level wasslightly decreased in the culture receiving supplemental tyrosine (datanot shown).

Example 2

Another series of cultures was prepared as described above and weresupplemented with independent feeds of a concentrated tyrosine feedsolution at 1 mM (control), 1.5 mM, 2 mM and 4 mM (negative control) todetermine an optimal tyrosine concentration in the cell culture medium.The first independent tyrosine feed was administered on day 7 ratherthan day 9 to maintain higher tyrosine levels earlier in the cellculture medium to avoid possible depletion. Independent tyrosine feedswere then given on subsequent feed events after day 7. Survivingcultures were carried for 28 days instead of 16 with additionalindependent tyrosine feeds on days 16, 18 and 21. Additional sampleswere taken on day 18 and daily from day 21 to 28. The amount ofconcentrated feed media was reduced to 7% (7 mls) provided on days 16and 18 and 5% (5 mls) on day 21.

Tyrosine additions targeting 1.5 mM, 2 mM and 4 mM starting on day 7 didnot show any additional benefit to performance compared to the 1 mMcontrol (FIGS. 3A to 3D). The 4 mM tyrosine feeds raised the pH as highas 7.45 but did not negatively affect VCD (data not shown), viability ortiter. However, high levels of tyrosine caused the culture to formclumps that stuck to the bottom of the flask due to precipitation. Spentmedia analysis showed that these cultures accumulated concentrations oftyrosine of between 8 to 9 mM, and that again, phenylalanine was notutilized (FIG. 3E and 3F). For those cultures carried beyond 16 days thetiter was 8.28 g/L and the Qp was maintained along with cultureviability at 28 days (FIGS. 3A to 3C). Supplying extra tyrosine startingon day 7 helped maintain higher tyrosine levels earlier in the cultureand avoided depletion (FIG. 3E). NH₄ ⁺ levels were maintained under 8 mMwhen tyrosine was first added on day 7 versus 10 mM on day 9 (data notshown).

Tyrosine depletion in production phase cell cultures resulted in a rapiddrop in viability, a decrease in Qp and a loss of titer even though thecultures were routinely fed with a concentrated feed medium containing amaximal amount of tyrosine. By delivering extra tyrosine using the abovedescribed independent feeding method, viability was prolonged, celldiameter increased and Qp maintained during the stationary phase of theculture resulting in significantly higher titers. The prolongedviability allowed for the extension of the culture duration which iscritical for enhancing productivity.

The benefits of having non-depleted tyrosine levels in the culture areevident from the above results. However, due to tyrosine's lowsolubility limitation, compounding more tyrosine into the concentratedfeed medium formulation was not a solution. Preparing a concentratedtyrosine feed solution and using it to provide independent additions oftyrosine to the cell culture medium was demonstrated to maintain a levelof tyrosine in the cell culture medium that was not achievable using theconcentrated feed medium and prevented depletion and the resultingdecline in cell viability, Qp and titer without disruption of theproduction phase of the culture. This method is production friendly andcan be readily implemented without any significant changes to aproduction facility.

Example 3

In this experiment, either tyrosine or tyrosine and cysteine wereremoved from the concentrated feed medium and provided as separatetyrosine and/or cysteine feeds.

Chinese hamster ovary (CHO) cells expressing a recombinant antibody wereinoculated at 1×10⁶ cells/mL into a defined serum-free culture mediumcontaining 0.98 g/L tyrosine and 0.5 g/L cysteine. The cultures weremaintained in 250 mL vented shake flasks with a 50 mL culture volume.The cultures were incubated at 36° C., 5% CO₂, 70% relative humidity,and 160 rpm on a platform having a 50 mm orbital diameter.

A 100 g/L L-tyrosine 2Na⁺2H₂O (SAFC Biosciences, Lenexa, Kans.)concentrated tyrosine feed solution was prepared in distilled H₂O. A 100g/L L-cysteine HCl (JT Baker/Avantor Performance Materials,Phillipsburg, N.J.) concentrated cysteine feed solution was prepared indistilled H₂O.

The culture was maintained for 13 days. Glucose was maintained at >2g/L.

On days 3, 6, and 8, the cells were fed: (a) a concentrated definedserum-free feed medium containing cysteine and sodium pyruvate butlacking tyrosine. In addition, 0.188 mL of the 100 g/L concentratedtyrosine feed solution was provided as an independent feed. (b) aconcentrated defined serum-free feed medium that lacked both tyrosineand cysteine but contained sodium pyruvate. In addition, 0.188 mL of the100 g/L concentrated tyrosine feed solution and 0.093 mL of 100 g/Lconcentrated cysteine feed solution were provided as independent feeds.

Samples were collected on days 3, 5, 6, 7, 8, 10, 11, 12 and 13. On eachsample day, viable cell density, viability and glucose concentrationwere determined Titer was determined on days 8, 10, 11, 12 and 13.

Viable cell density (VCD) and viability were determined by GuavaViacount assay (Millipore Billerica, Mass.) and metabolites (glucose)using reagent kits from Polymedco Inc. (Cortlandt Manor, N.Y.). Titerwas measured by Protein A HPLC analysis using affinity chromatographywhere Protein A was immobilized on a column support. At neutral pH,antibody molecules were bound to the Protein A through the Fc regionwhile host-cell proteins, conditioned media components and buffer wereeluted from the column in the flow-through. Captured antibodies wereeluted at acidic pH and detected by UV absorbance at 280 nm. Acalibration curve was derived from an antibody standard and thecorresponding peak areas using linear regression analysis.Concentrations of the antibody in the test samples were then calculatedfrom the calibration curve. Lactate was measured using a YSI Selectanalyzer (YSI, Inc., Yellow Springs, Ohio).

Growth, viability and antibody productivity were not diminished as aresult of removing tyrosine and cysteine from the feed medium anddelivering them as a separate concentrated feeds (FIG. 1A-C).

Example 4

It is desirable from a process operations perspective to keep the numberof feed steps to a minimum. Combining the concentrated cysteine andtyrosine feeds into a single concentrated feed solution would minimizethe number of feed steps while maintaining the desirable flexibility ofindependent tyrosine and cysteine feeds. However, cysteine in itsreduced form is not soluble with tyrosine due to the high pH of thetyrosine solution. To overcome this incompatibility, the oxidized form,cystine, was used. Cystine and tyrosine are both readily soluble atpH >8 and solubility is high at pH >10. Both cystine and tyrosine can bedissolved in a sodium hydroxide solution (for example 0.1N NaOH) and theresulting solution has the added benefit of not requiring subsequentviral inactivating treatments such as pasteurization or viralfiltration.

A comparison was made of independent tyrosine and cysteine feeds with anindependent feed of a combined tyrosine and cystine stock.

CHO cells expressing a recombinant antibody were inoculated at 9×10⁵cells/mL into a defined serum-free culture medium containing 0.98 g/Ltyrosine and 0.5 g/L cysteine. The cultures were maintained in 1 Lbioreactors with an 800 mL initial working volume. Temperature wasmaintained at 36° C., agitation rate was 290 rpm, dissolved oxygen wasmaintained at 30% by sparging, pH was maintained at 7.0 and wascontrolled by CO2 and sodium carbonate addition as required. Glucose wasmaintained at >2 g/L.

In addition to the concentrated tyrosine and cysteine feed solutionsdescribed above, a 100 g/L L-tyrosine 2Na⁺2H₂O (SAFC Bioscience), 33.5g/L L-cystine (Sigma-Aldrich), 0.1N NaOH (JT Baker/Avantor PerformanceMaterials) feed solution was prepared.

On days 3, 6, and 8, the cells were fed: (a) a concentrated definedserum-free feed medium lacking tyrosine and cysteine. In addition, 1.9mL of the 100 g/L concentrated cysteine feed solution and 3.9 mL of the100 g/L concentrated tyrosine feed solution were provided as independentfeeds. (b) the same concentrated defined serum-free feed medium lackingtyrosine and cysteine. In addition, 3.9 mL of the 33.5 g/L cystine and100 g/L tyrosine concentrated feed solution was provided as anindependent feed.

Feeding a concentrated cysteine feed solution and a concentratedtyrosine feed solution separately or feeding a combined concentratedcystine and tyrosine feed solution resulted in equivalent cell cultureperformance; therefore, the two nutrients can be combined into a singleindependent feed solution to simplify the feed operations withoutdiminishing the performance of the culture (FIG. 2A-D).

What is claimed is:
 1. A method of culturing Chinese Hamster Ovary (CHO)cells expressing a recombinant protein, comprising growing the CHO cellsin a defined serum-free culture medium during a growth phase andmaintaining the CHO cells in the cell culture medium during a productionphase by supplementing the cell culture with a concentrated serum-freedefined feed medium containing tyrosine and further supplementing thecell culture with one or more independent tyrosine feeds, wherein theindependent tyrosine feed provides at least about 1 mM to at least about2 mM tyrosine at each feed, wherein viability was prolonged, specificproductivity was maintained, and titer was improved compared to CHOcells not receiving independent tyrosine feeds.
 2. The method accordingto claim 1, wherein the independent tyrosine feed provides at leastabout 1 mM tyrosine.
 3. The method according to claim 2, wherein theindependent tyrosine feed provides at least about 1.38 mM tyrosine. 4.The method according to claim 1, wherein the concentration of tyrosinein the cell culture medium does not exceed 8 mM.
 5. The method accordingto claim 1, wherein the independent tyrosine feed begins at least by day5 of the production phase.
 6. The method according to claim 1, whereinthe independent tyrosine feed begins on day 3 of the production phase.7. The method according to claim 1, wherein the independent tyrosinefeeds begin on day
 7. 8. The method according to claim 1, wherein theindependent tyrosine feed begins prior to the production phase.
 9. Themethod according to claim 1, wherein the independent tyrosine feed ismade concurrently with the feed of the concentrated serum-free definedfeed medium.
 10. The method according to claim 1, wherein theindependent tyrosine feed is not concurrent with the feed of theconcentrated serum-free defined feed medium.
 11. The method according toclaim 1, wherein the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine.